Improved Specificity of Newborn Screening for Congenital Adrenal Hyperplasia by Second-Tier Steroid Profiling Using Tandem Mass Spectrometry

Jean M. Lacey, Carla Z. Minutti, Mark J. Magera, Angela L. Tauscher, Bruno Casetta, Mark McCann, James Lymp, Si Houn Hahn, Piero Rinaldo, Dietrich Matern

Research output: Contribution to journalArticlepeer-review

167 Scopus citations


Background: Newborn screening for congenital adrenal hyperplasia (CAH) involves measurement of 17α-hydroxyprogesterone (17-OHP), usually by immunoassay. Because this testing has been characterized by high false-positive rates, we developed a steroid profiling method that uses liquid chromatography-tandem mass spectrometry (LC-MS/MS) to measure 17-OHP, androstenedione, and cortisol simultaneously in blood spots. Methods: Whole blood was eluted from a 4.8-mm (3/16-inch) dried-blood spot by an aqueous solution containing the deuterium-labeled internal standard d 8-17-OHP. 17-OHP, androstenedione, and cortisol were extracted into diethyl ether, which was subsequently evaporated and the residue dissolved in LC mobile phase. This extract was injected into a LC-MS/MS equipped with pneumatically assisted electrospray. The steroids were quantified in the selected-reaction monitoring mode by use of peak areas in reference to the stable-isotope-labeled internal standard. We analyzed 857 newborn blood spots, including 14 blood spots of confirmed CAH cases and 101 of false-positive cases by conventional screening. Results: Intra- and interassay CVs for 17-OHP were 7.2-20% and 3.9-18%, respectively, at concentrations of 2, 30, and 50 μg/L. At a cutoff for 17-OHP of 12.5 μg/L and a cutoff of 3.75 for the sum of peak areas for 17-OHP and androstenedione divided by the peak area for cortisol, 86 of the 101 false-positive samples were within reference values by LC-MS/MS, whereas the 742 normal and 14 true-positive results obtained by conventional screening were correctly classified. Conclusion: Steroid profiling in blood spots can identify false-positive results obtained by conventional newborn screening for CAH.

Original languageEnglish (US)
Pages (from-to)621-625
Number of pages5
JournalClinical chemistry
Issue number3
StatePublished - Mar 2004

ASJC Scopus subject areas

  • Clinical Biochemistry
  • Biochemistry, medical


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